Embryonic stem (ES) cells isolated from the inner cell mass of murine blostocysts are pluripotent, capable of indefinite symmetric cell division, and can generate chimeric animals. The recent isolation of human ES cells holds great promise for the treatment of a variety of degenerative disorders including, but not limited to, Parkinson's disease and diabetes. The homeodomain transcription factors, Oct3/4 and Nanog, are believed to play critical roles in sustaining ES cell pluripotency. Surprisingly, targets of Nanog are completely unknown and few Oct3/4 targets have been proposed. Thus, the molecular mechanisms that repress differentiation of ES cells and promote self-renewal are poorly defined. We have developed a novel approach for identifying complex metazoan regulons called SACO (Serial Analysis of Chromatin Occupancy), which combines chromatin immunoprecipitation with a modification of Long SAGE. We will use SACO to identify the entire complement of Nanog and Oct3/4 genomic targets in mouse ES cells. Such knowledge would not only aid efforts to characterize mechanisms that govern cell-fate commitment of stem cells, but could also enable the indefinite propagation of existing human stem cell lines. Insight into these transcriptional networks could also lead to the generation of pluripotent stem cells from adult tissues or cells. Our studies are designed to provide a complete definition of Nanog and Oct3/4 targets in pluripotent stem cells. By characterizing the regulation of the corresponding transcripts, we expect to characterize the molecular pathways that control self-renewal and pluripotency of ES cells. The microarrays representing novel Nanog and Oct3/4 driven transcripts and the catalog of genomic binding sites will be provided to the stem cell research community. The creation of microarrays representing novel Nanog and Oct3/4 targets will facilitate high-throughput analysis of their expression during early embryogenesis and their regulation by pathways that regulate differentiation and pluripotency. This set of studies will represent the most comprehensive analysis to date of the regulation of transcription factor binding in any metazoan system.